The long-term goal of this proposal is to understand the role of lipid-protein interactions in the synthesis, assembly, structure and function of membrane proteins. Through the use of a combined molecular genetic and biochemical approach we have established a specific role for the phospholipid phosphatidylethanolamine in determining the native topological organization of the transmembrane domains of a subset of membrane proteins, namely secondary transporters of sugars and amino acids in Escherichia coli. IVIost dramatic was the observation that large topological reorganization in transmembrane domain orientation can occur post-assembly of a membrane protein by changes in the lipid environment. From these studies a set of rules is evolving in which short-term cooperative charge interactions between membrane lipids and proteins coupled with long-term interactions between transmembrane domains are important determinants of final topological organization of the above proteins. The four aims of this proposal are designed to provide more precise molecular details on how lipid-protein interactions influence membrane protein structure, better define the rules, cellular factors and mechanism of lipid-dependent topogenesis and establish the generality of lipid-protein interactions in determining final topological organization. In Aim 1 the details of changes in organization of lactose permease as a function of lipid environment will be established using high-resolution X-ray crystallography. In Aim 2 host cell processes that act in concert with the lipid environment in establishing native topology will be identified. In addition the list of topogenic signals within protein sequences will be broadened and refined. In Aim 3 protein sequences and the properties of the lipid environment that determine final membrane protein topology and changes in topology post-assembly of membrane proteins will be further detailed using an in vitro reconstituted system. In Aim 4 the question of generality ef lipid-dependent topogenesis will be addressed first for additional pound. coli proteins and then extended to yeast and higher eukaryotic membrane proteins. Overall these studies will place lipid-protein interactions as determinants of protein structure on a firmer mechanistic basis. More precisely defining the rules governing membrane protein folding will provide important information on diseases such as cystic fibrosis, all forms of dementia, scapies, and diabetes that involve protein misfolding events involving lipid- protein interactions.